Significance of metabolic activation of lumisterol in the skin

皮肤中光甾醇代谢激活的意义

基本信息

批准号：
9756150
负责人：
ANDRZEJ T SLOMINSKI
金额：
$ 32.85万
依托单位：
UNIVERSITY OF ALABAMA AT BIRMINGHAM
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-09-01 至 2022-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9756150
关键词：
7-dehydrocholesterol Adrenal Glands Binding Biochemical Pathway Biochemistry Bioinformatics Biological Blood Circulation C10 CYP11A1 gene Cells Chemistry Chinese Hamster Ovary Cell Cholecalciferol Cholesterol Cleaved cell Computer Simulation Coupled Crystallization Cutaneous DNA Damage Data Docking Dose Enzymes Epidermis Evaluation Exposure to Follow-Up Studies Gene Expression Gene Silencing Genes Genetic Complementation Test Genetic Transcription Genomics Gonadal structure Homeostasis Human Human body Intoxication Investigation Ligand Binding Domain Ligands Measures Mediating Metabolic Metabolic Activation Methods Modeling Molecular Molecular Biology Molecular Conformation Mus Organ Organ Culture Techniques Outcome Oxidative Stress Pathology Pathway interactions Pharmacology Phenotype Physiological Placenta Play Pregnenolone Process Production Recombinants Regulation Reporter Role Sampling Scheme Serum Side Signal Transduction Skin Skin Physiology Stereoisomer Steroid biosynthesis Steroids Structure System Techniques Testing Tetanus Helper Peptide Transactivation UVB induced Ultraviolet B Radiation absorption base design genetically modified cells in vivo interest keratinocyte keratinocyte differentiation molecular modeling multidisciplinary novel orphan nuclear receptor ROR-gamma photoprotection predictive modeling prevent programs receptor simulation skin barrier stressor transcriptome sequencing whole genome

项目摘要

UVB breaks the B-ring of 7-dehydrocholesterol leading to the formation of pre-vitamin D3 that either undergoes thermal isomerization to D3 or photoisomerization to lumisterol (L3) in a 9β,10α-configuration. L3 is the major photoisomer formed after prolonged exposure of the skin to UVB energy, in a process that is irreversible. L3 has been considered to be metabolically and biologically inactive with its formation explaining why UVB induced cutaneous production of pre-D3 does not lead to systemic intoxication by D3. Recently we found that L3 is metabolized by CYP11A1 to 22(OH)L3, 24(OH)L3, 20,22(OH)2D3 and pregnalumisterol (pL) [collectively called (OH)nL3]. Uning LC/qTOF-MS we demonstrated the presence of L3 in human serum at levels higher than D3, and detectable levels of (OH)nL3 in the epidermis and serum. Furthermore, we found that (OH)nL3 inhibits the proliferation of human epidermal keratinocytes and that 20(OH)L3 stimulates the expression of genes associated with keratinocyte differentiation and protection against oxidative stress. Our preliminary molecular modeling predicts that the major (OH)nL3 products can act on RORα and RORγ showing favorable docking scores in the ligand binding domain (LBD) of the receptors, similar to known natural ligands. Supporting these predictions we found that 20(OH)L3 inhibits RORγ and RORα transactivation activities in a Tet-on CHO cell reporter system, as well as reducing (RORE)-LUC reporter activity in skin cells. Thus, we have discovered a new lumisterogenic biochemical pathway that is biologically relevant. To investigate its role in skin physiology and pathology we formulate the hypothesis that skin-derived L3 is enzymatically activated generating (OH)nL3 compounds which regulate epidermal barrier and photoprotective functions through interaction with RORα and RORγ. This hypothesis will be tested via three aims. 1. Defining RORα and RORγ as functional receptors for novel hydroxylumisterol derivatives ((OH)nL3); 2. Defining the phenotypic activities of (OH)nL3 in cultured epidermal keratinocytes and in human skin histocultured ex vivo; 3. Defining the relative roles of RORα and RORγ in (OH)nL3-mediated regulation of the differentiation program and protective mechanisms against UVB radiation in the epidermis. Our highly mechanistic strategy combines techniques of primary epidermal and organ cultures, in silico methods, cell-based transcriptional studies coupled to the LBD of RORα/γ, the use of genetically modified cells, whole genome RNA sequencing with bioinformatics, and the techniques of biochemistry, chemistry and molecular biology. This proposal is a comprehensive, multi- disciplinary and state-of-the-art investigation. Its significance encompasses defining a role for a previously unrecognized pathway of activation of an important UV photoproduct, L3, in skin physiology and pathology. The practical outcome of the realization of aims 1-3 would be the use of the most potent (OH)nL3 as agent(s) that either enhance the epidermal barrier or prevent or reverse damage inflicted by noxious stressors and UVB. Establishing roles for RORs in these processes will define them as druggable pharmacological targets.

UVB 破坏 7-脱氢胆固醇的 B 环，导致前维生素 D3 的形成，主要是热异构化为 D3 或光异构化为 9β,10α-L3 构型。皮肤长时间暴露于 UVB 能量后形成光异构体，该过程是不可逆的 L3。被认为在代谢和生物学上不活跃，其形成解释了为什么 UVB 最近我们发现，诱导皮肤产生前 D3 不会导致 D3 全身中毒。 L3 由 CYP11A1 代谢为 22(OH)L3、24(OH)L3、20,22(OH)2D3 和 pregnalumisterol (pL) [统称为称为 (OH)nL3]，通过 LC/qTOF-MS，我们证明了人血清中 L3 的存在水平较高。比 D3 更高，并且表皮和血清中可检测到 (OH)nL3 水平。此外，我们发现 (OH)nL3。抑制人表皮角质形成细胞的增殖，20(OH)L3 刺激我们的初步研究与角质形成细胞分化和氧化应激保护相关的基因。分子模型预测主要 (OH)nL3 产物可以作用于 RORα 和 RORγ，显示出良好的效果受体配体结合域（LBD）中的对接分数，类似于已知的天然配体。我们发现 20(OH)L3 抑制 RORγ 和 RORα 反式激活活性，支持这些预测。 Tet-on CHO 细胞报告系统，以及减少皮肤细胞中的 (RORE)-LUC 报告活性。发现了一种新的发光生化途径，该途径具有生物学相关性，以研究其作用。在皮肤生理学和病理学中，我们提出了皮肤来源的 L3 被酶促激活的假设生成 (OH)nL3 化合物，通过调节表皮屏障和光保护功能与 RORα 和 RORγ 的相互作用该假设将通过三个目标进行检验 1. 定义 RORα 和 RORγ。作为新型羟基米甾醇衍生物 ((OH)nL3) 的功能受体；2. 定义表型活性 3. 定义相对值； RORα和RORγ在(OH)nL3介导的分化程序和保护性调节中的作用我们的高度机械化策略结合了以下技术：原代表皮和器官培养物、计算机方法、与 LBD 耦合的基于细胞的转录研究 RORα/γ、转基因细胞的使用、生物信息学的全基因组 RNA 测序以及该提案是一项综合性、多学科的生物化学、化学和分子生物学技术。其重要性包括为先前的人员定义角色。皮肤生理学和病理学中重要的紫外线光产物 L3 的激活途径尚未被认识。实现目标 1-3 的实际结果是使用最有效的 (OH)nL3 作为试剂增强表皮屏障或预防或逆转有害压力源造成的损害 UVB。确定 ROR 在这些过程中的作用将把它们定义为可成药的药理学靶点。